This proposal represents our continuing efforts to understand the genetics, cellular basis and somatic evolution of protective polysaccharide (PS) antibody (Ab) repertoires. We will focus upon Ab repertoires specific for Streptococcus pneumoniae polysaccharide serotypes 14 (PPS 14) and 23F (PPS 23F) and the Haemophilus influenzae type b capsular polysaccharide (Hib PS). Preliminary studies lead us to hypothesize that the primary PPS B cell repertoire is of low affinity and relies upon hypermutation, driven by conjugate vaccination, to generate a high affinity, protective response. The molecular ontogeny of the PPS Ab repertoire will be examined by isolating PPS-specific B cells from infants following immunization with PPS-protein conjugate vaccines. The variable (V) region genes used by individual PPS-specific B cells or their clonal products will be sequenced and affinity analyses will be performed on monoclonal Fab fragments. Selected Fab fragments will be converted to full-length recombinant Abs in order to evaluate opsonophagocytic activity. These studies will identify the V genes used by infants in response to PPS 14 and PPS 23F, document the extent to which somatic hypermutation and/or V region shifts occur in the maturation of the response and determine the functional consequences of this mutation. Our in vitro studies indicate that IgH allelic polymorphisms can dramatically influence PS Ab function. Accordingly, we will perform vaccination and B cell cloning studies in infants and adults to test the hypothesis that individuals utilizing the V3-23*03 allele will produce higher quality canonical Hib PS Abs and PPS 23F Abs than individuals using the V3-23*01 allele. Little is known about the specificity repertoire of peripheral blood IgM memory B cells or their role in PS immunity. We will identify the V region repertoire of PS-specific IgM 'memory'B cells isolated from vaccinated adults. V gene usage and mutation will be evaluated in individual IgM memory B cells or expanded clones, and PS binding studies will performed on Fab fragments derived from these clones. We will use a stable isotope in vivo labeling method to determine the DMA synthetic rates (half lives) of IgM memory B cells and other blood B cell subsets both before and after vaccination to determine whether vaccination induces DNA synthesis (clonal proliferation) in these B cells. These studies address clinically relevant topics. They will identify genetic determinants of Ab efficacy and disease susceptibility, elucidate the cells and processes involved in the immune response to pediatric vaccines, and delineate the mechanisms underlying immunity to encapsulated pathogens. This research will deepen our understanding of the human immune system and may contribute toward the design of better vaccines.